Gastric SMARCA4-deficient undifferentiated tumor (SMARCA4-UT): a clinicopathological analysis of four rare cases

Background SMARCA4, as one of the subunits of the SWI/SNF chromatin remodeling complex, drives SMARCA4-deficient tumors. Gastric SMARCA4-deficient tumors may include gastric SMARCA4-deficient carcinoma and gastric SMARCA4-deficient undifferentiated tumor (SMARCA4-UT). Gastric SMARCA4-UT is rare and challenging to diagnose in clinical practice. The present report aims to provide insight into the clinicopathological characteristics and genetic alterations of gastric SMARCA4-UTs. Results We retrospectively reported four rare cases of gastric SMARCA4-UTs. All four cases were male, aged between 61 and 82 years. These tumors presented as ulcerated and transmural masses with infiltration, staged as TNM IV in cases 1, 2 and 4, and TNM IIIA in case 3. Pathologically, four cases presented solid architecture with undifferentiated morphology. Cases 2 and 3 showed focal necrosis and focal rhabdoid morphology. Immunohistochemical staining showed negative expression of epithelial markers and deficient expression of SMARCA4. Furthermore, positivity for Syn (cases 1, 2 and 3) and SALL4 (cases 1 and 2) were observed. Mutant p53 expression occurred in four cases, resulting in strong and diffuse staining of p53 expression in cases 1, 2 and 4, and complete loss in case 3. The Ki67 proliferative index exceeded 80%. 25% (1/4, case 4) of cases had mismatch repair deficiency (dMMR). Two available cases (cases 1 and 3) were detected with SMRACA4 gene alterations. The response to neoadjuvant therapy was ineffective in case 1. Conclusions Gastric SMARCA4-UT is a rare entity of gastric cancer with a poor prognosis, predominantly occurs in male patients. The tumors are typically diagnosed at advanced stages and shows a solid architecture with undifferentiated morphology. Negative expression of epithelial markers and complete loss of SMARCA4 immunoexpression are emerging as a useful diagnostic tool for rare gastric SMARCA4-UTs.


Background
Gastric cancer (GC) is one of the most common malignancies globally and has poor outcome, especially in Asia [1,2].According to the fifth edition of the World Health Organization (WHO) classification of digestive tumors, undifferentiated gastric carcinomas are rare highly aggressive tumors showing no specific cytological or architectural type of differentiation [3].Some undifferentiated gastric carcinomas may exhibit rhabdoid features, comprising 0.1-0.3% of poorly differentiated adenocarcinoma, and 5.0-5.6% of solid adenocarcinomas as reported in literature [4,5].The undifferentiated phenotype is probably driven by various components of the switch/sucrose non-fermenting (SWI/SNF) chromatinremodeling complex in some cases [3,6,7].
Immunohistochemical staining for SMARCA4 is useful to identify SMARCA4-deficient tumors.Gastric SMARCA4-deficient tumor may include gastric SMARCA4-deficient carcinoma and gastric SMARCA4-UT.Gastric SMARCA4-deficient carcinoma could be differentiated from gastric SMARCA4-UT based on gland architecture, cellular cohesion and diffuse strong keratin expression.Furthermore, gastric SMARCA4-UT is typical loss of epithelial differentiation (negative expression of epithelial markers such as pancytokeratin (PCK) and epithelial membrane antigen (EMA)), whereas gastric SMARCA4-deficient undifferentiated carcinoma reveals variable expression of epithelial markers [13].
Gastric SMARCA4-UT is a rare entity of gastric tumors.Due to its rarity, the clinicopathological significance and molecular features are limited.Herein, we present four rare cases of gastric SMARCA4-UTs and provide insight into the clinicopathological characteristics and genetic alterations of these highly aggressive malignant tumors.

Clinical features
Four cases diagnosed with gastric SMARCA4-UTs were all male, aged between 61 and 82 years.These tumors presented with ulcerated and advanced tumors, staged as tumor-node-metastasis (TNM) IV in cases 1, 2 and 4, and TNM IIIA in case 3. Tumor size varied from 2 to 6 cm in maximum diameter.Detailed clinical features are summarized in Table 1.
A 61-year-old male patient (case 1) who presented with dysphagia, abdominal pain and bloating for approximately one month was admitted to our hospital.A computed tomography (CT) scan of the abdomen showed a 6 cm irregular thickened tumor in the cardia, fundus and lesser curvature of the stomach (Fig. 1A).There were multiple enlarged lymph nodes in the ligamenta hepatogastricum, portacaval space, mesentery and abdominal aorta, which were partially fused, and a nodule in right posterior lobe of the liver.Gastroscopy showed a large ulcerative tumor in the cardia of the stomach, and a biopsy was performed.The clinical TNM stage was TNM VI.The patient received neoadjuvant therapy with a combination of chemotherapy (irinotecan and cisplatin) and sintilimab (a PD-1 inhibitor).A partial response (PR) was achieved after two cycles.The patient received continued treatment after six cycles with progressive disease (PD).Subsequently, this patient received the second line chemotherapy with albumin paclitaxel combined with tigio, and a PR was achieved after three cycles in clinical assessment.And then, surgical resection was performed.
A 64-year-old male patient (case 2) presented with intermittent hematemesis and abdominal pain for three months.The CT scan of the abdomen and gastroscopy showed a 2 cm thickened lesion in lower esophagus and cardia of the stomach (Fig. 2A-B).A biopsy was performed.Besides, the CT scan showed multiple nodules in liver, indicating metastases of the liver; Additionally, lymph nodes in hepatogastric ligaments and portal space were enlarged and partially fused.The clinical TNM stage was TNM VI.
A 61-year-old male patient (case 3) presented with upper abdominal discomfort with bloating for approximately one month and was admitted to the local hospital.Gastroscopy was performed and showed a 4 cm mass in the lateral posterior wall of the stomach.Gastrectomy was performed at the local hospital.The pathological TNM stage was T4aN1M0, IIIA.The patient received chemotherapy after surgical resection.
An 82-year-old male patient (case 4) presented with abdominal pain for approximately one month and was admitted to the local hospital.A CT scan of the abdomen

Pathological features
Cases 3 and 4 were admitted and treated at the local hospital, and pathological consultation was submitted to our hospital.All four cases had similar histological characteristics.Morphological observation showed undifferentiated tumor cells that formed a solid architecture without tubular glandular formation (Figs. 1B-C, 2C-E and 3A-B, and 4A-B).The large-to medium-sized tumor cells were epithelioid ovoid or polygonal cells with abundant cytoplasm.Round, pleomorphic nuclei with prominent nucleoli were large and irregular.Mitoses were frequent.Cases 2 and 3 showed focal necrosis (Figs.2D and 3A) and focal rhabdoid morphology (Figs.2E and 3C).The tumor regression grade (TRG) for case 1 with neoadjuvant therapy was TRG3 (without an obvious response to neoadjuvant treatment) (Fig. 1C), while the lymph nodes showed a response to neoadjuvant therapy (Fig. 1D).
The tumor cells of case 4 was negative expression of PCK, EMA and CK7.The present patient had mismatch repair deficiency (dMMR), with deficient expression of MLH1 and PMS2, and retained expression of MSH2 and MSH6 (Fig. 4).The expression of p53 was strong and diffuse staining, and the Ki67 proliferative index was approximately 80%.

Molecular analysis
In situ hybridization for EBER was negative in available cases (cases 1 and 2).The next-generation sequencing (NGS) of case 1 confirmed deletion mutations in SMARCA4, RAD51 and TSC2, and the tumor mutation burden (TMB) was 0.96 mutations/Mb.The NGS of case

Discussion
SMARCA4 is associated with progression and poor prognosis in gastric cancer [7,25].Inactivation of SMARCA4 rarely occurs in classic glandular gastric cancer as a driver molecular event and is more likely to occur in gastric cancer with solid and poorly differentiated and undifferentiated morphology [6,13,16,26], and loss of SMARCA4 is associated with adverse clinical characteristics [11,12].Patients with undifferentiated carcinoma of the gastrointestinal (GI) tract exhibiting loss of SMARCA4 expression demonstrated significantly poorer overall survival (p = 0.028) and disease-free survival (p = 0.006) compared to those with SMARCA4 expression [7].In the present report, all four cases were male aged ranged from 61 to 82 years, presented as large ulcerated and transmural masses with infiltration and were staged as TNM IV in cases 1, 2 and 4, and TNM IIIA in case 3 at the time of diagnosis.Tumors presented solid architecture with undifferentiated morphology and showed complete loss of SMARCA4 (BRG1) expression.In addition, 50% (2/4, cases 2 and 3) showed focal necrosis and rhabdoid morphology, comprised less than 1% of the tumor cells.Rhabdoid cells could be an important diagnostic clue.Immunohistochemical staining for SMARCA4 (BRG1) should be performed in gastric cancers with solid architecture and undifferentiated components, especially those with rhabdoid morphology.
Gastric SWI/SNF complex-deficient tumors may have similar histopathological features with undifferentiated or poorly differentiated morphology [7].Agaimy et al. reported that most gastric undifferentiated/rhabdoid carcinoma cases (12/13) in the GI tract lack expression of at least 1 of the 4 switch/sucrose nonfermenting (SWI/SNF) complex subunits (SMARCB1, SMARCA2, SMARCA4, and ARID1A) [6].In 477 adenocarcinomas of the stomach and gastroesophageal junction, 32% of cases demonstrated aberrant expression of the SWI/SNF complex, and SWI/SNF aberration emerged as an independent negative prognostic factor for overall survival [14].However, SMARCB1, SMARCA2 and ARID1A were not detected by NGS in the present two cases.D-MMR is a good independent prognostic factor in advanced gastric cancer [34].The SWI/SNF mutations are enriched in microsatellite instability (MSI) genotype [14].SMARCA4 mutation was detected in 48.84% of 43 cases with d-MMR GC patients by NGS [35].SWI/SNF loss is superimposed on mismatch repair deficiency in a subset of cases [6].Sasaki T et al. demonstrated that 52.0% (13/25) of solid-type poorly differentiated adenocarcinoma had deficient mismatch repair d-MMR [12].In the present study, 25% (1/4) of cases had d-MMR.
A group of tumors with similar morphologic features should be excluded before diagnosing gastric SMARCA4-UT.The differential diagnosis for gastric SMARCA4-UT includes gastric undifferentiated or poorly differentiated carcinoma with SMARCA4 deficiency, neuroendocrine carcinoma (NEC), EBV-associated carcinoma with lymphoid stroma, lymphomas (including anaplastic large cell lymphoma), melanoma, germ cell neoplasms, NUT-midline carcinoma and so on.Gastric SMARCA4deficient carcinoma could be distinguished from gastric SMARCA4-UT by gland architecture, cellular cohesion, and diffuse strong keratin expression.Decreased expression of PCK was observed in 58.6% (17/29) of gastric SMARCA4-deficient undifferentiated carcinomas [13].SALL4 and CD34 were positivity in some gastric SMARCA4-UTs.NEC often diffusely expresses the neuroendocrine markers CgA, Syn and CD56, and tumor cells express epithelial markers, which are helpful for the diagnosis of NEC with large-cell and/or rhabdoid features.The neuroendocrine marker Syn was positively expressed in cases 1, 2 and 3, however, none of the cases exhibited co-expression of other neuroendocrine markers CgA and CD56.Foci of abrupt squamous differentiation can often be identified in NUT-midline carcinoma, and tumor cells can be shown to harbor BRD-NUT fusions and NUT-positive expression.
Further analysis showed that patients with SMARCA4altered GC did not benefit from chemotherapy in stages II and III (p = 0.623 and 0.678).In patients with stage III disease who received chemotherapy, SMARCA4-altered GC remained a significant unfavorable prognostic factor (median survival 14 versus 26 months, p = 0.002) [29].Another study demonstrated that the response to preoperative chemotherapy of SWI/SNF-aberrant gastric carcinomas were TRG2 (22%, 8/41) and TRG3 (78%, 32/41) [11].SMARCA4-altered gastric cancers may do not benefit from chemotherapy and had poor outcomes.Two patients localized at the gastroesophageal junction received neoadjuvant chemotherapy and showed no response (TRG3), showing very adverse clinical characteristics and poor survival [11].BRG1-associated expression of 9-27 and IFI-27 is involved in cisplatin resistance in gastric cancer cells [36].In the present study, case 1 received chemotherapy and a PD-1 inhibitor before surgical treatment, and the response to conventional chemotherapy was ineffective.The tumor showed TRG3 in response to neoadjuvant therapy.Future treatments with target agents such as inhibitors against enhancer of zeste homolog 2 (EZH2) or histone deactylase, may prove even more effective [37,38].

Conclusions
The present four rare gastric SMARCA4-UTs were aggressive malignancies, occurred in male patients, with advanced stages.These tumors showed solid architecture with undifferentiated morphology.Immunohistochemical staining was negative expression of epithelial markers and complete loss of SMARCA4.Further studies with larger sample size are needed.

Case selection
The data of four cases of gastric SMARCA4-UT were reviewed from the database of the Department of Pathology, West China Hospital, Sichuan University between 2019 and 2022.We retrospectively recorded clinicopathological and demographic characteristics.Clinical and radiographic features were obtained from patients' medical records and follow-up.

Fig. 1
Fig.1Radiological, histopathological and immunohistochemical characteristics of case 1.A CT scan of the abdomen showed a large irregular thickened area of the cardia, fundus and lesser curvature of the stomach before neoadjuvant therapy(A).The tumor showed a solid architecture with undifferentiated morphology before (B, magnification x200) and after neoadjuvant therapy (C, magnification x200).There was no obvious response to neoadjuvant therapy (TRG3) (C, magnification x200).However, the lymph nodes showed a response to neoadjuvant therapy (D, magnification x10).Both biopsy and surgical resection specimen showed similar immunohistochemical staining.BRG1 was deficient expression in the nucleus of tumor cells.Both PCK and EMA were negative expression.The Ki67 proliferation index was approximately 80%.SALL4 and Syn were positively stained.P53 was positively expressed.(Magnification x200)

Fig. 2
Fig. 2 Gastroscopic, radiological, histopathological and immunohistochemical characteristics of case 2. Gastroscopy (A, arrow) and CT scan (B, arrow) showed a thickened lesion in lower esophagus and cardia of the stomach.The tumor showed a solid architecture with undifferentiated morphology (C, magnification x40) with focal necrosis (D, arrow, magnification x200).Partial tumor cells showed a rhabdoid morphology (E, magnification x200).BRG1 was deficient expression.Immunohistochemical staining showed negativity for both PCK and EMA, diffuse positivity for SALL4, and partial positivity for Syn.P53 was positively expressed.The Ki67 proliferation index was approximately 90%.(Magnification x200)

Fig. 3
Fig. 3 Histopathological and immunohistochemical characteristics of case 3. The tumor showed a solid architecture with comedonecrosis (A, arrow).High-power view showed undifferentiated tumor cells with poor cohesion (B, magnification x200).Partial tumor cells showed a rhabdoid morphology (C, magnification x400).BRG1 was deficient expression.PCK was negative expression.Expression of p53 was completely loss.CD34 and Syn were positively stained.The Ki67 proliferation index was approximately 80%.(Magnification x200)

Fig. 4
Fig. 4 Histopathological and immunohistochemical characteristics of case 4. A low-power view (A, magnification x40) and high-power (B, magnification x400) of the tumor showed a solid pattern with undifferentiated morphology.Immunohistochemical staining showed negative expression of CK7, deficient expression of BRG1 and positive expression of p53.This case showed mismatch repair deficiency (dMMR), with deficient expression of MLH1 and PMS2, and retained expression of MSH2 and MSH6 (Magnification x200)